It is required to impart vacuum resistance to the sample as the sample is in a vacuum and conductivity required to obtain an image to the sample in order to perform the observation of a sample by a scanning electron microscope and a transmission electron microscope.
The preparation of the sample for a scanning electron microscope is performed by removing the moisture from the sample through vacuum drying in advance, then depositing a conductive material (platinum, carbon, gold, palladium, osmium, or the like) on the sample in order to increase the generation efficiency of a secondary electron by imparting conductivity to the sample, and coating the surface of the sample by a means of a sputtering or the like.
Such a pretreatment is not required for those that are conductive and resistant to a vacuum, such as a metal or a semiconductor, but those that are not conductive are required to be coated with a conductive film formed of a conductive material. In addition, those that are inferior in vacuum resistance or the like, namely those that are deformed by vacuum drying or irradiation with an electron beam in a vacuum at the time of the electron microscopic observation are also required to be coated with a conductive film formed of a conductive material.
A biological sample is often required to be subjected to vacuum drying in advance since it contains a great amount of moisture. Hence, the surface shape of the sample is significantly deformed in some cases and it is difficult to observe it by an electron microscope in the living state.
It is possible to observe the water-containing state of a wet sample such as a gel-like substance or food at room temperature by using a low-vacuum SEM and a cryo-SEM, an environmental controlled SEM (ESEM), or the like. According to these methods, it is possible to observe not only a wet sample but also a sample as it is untreated. However, it is required to put the sample in a high vacuum in order to observe at a high magnification, and thus the sample is required to be resistant to a vacuum or conductive. Hence, it is difficult to obtain an image in the living state at a high magnification even in the observation of a biological sample.
In recent years, a method for an electron microscopic observation using an ionic liquid has been proposed. It is described that a wet sample is observed by using an ionic liquid and a SEM in Patent Literature 1 and Non Patent Literatures 1 and 2. The application of the ionic liquid to a cell is also described in Non Patent Literature 2. In addition, a method to use the technique of an ionic liquid for the transmission electron microscopic observation is disclosed in Patent Literature 2.